1. The Field of the Invention
The present invention relates to methods and apparatus for the isoelectric focusing of amphoteric substances. More particularly, the present invention is directed to the techniques for separating biological materials through the use of isoelectric focusing processes which enhance the separation characteristics of amphoteric biological substances and provide for efficient removal of heat generated during the isoelectric focusing process.
2. The Background of the Invention
Numerous areas of modern biology and genetic engineering depend on the availability of large quantities of high purity proteins. Currently available methods of protein purification include many kinds of chromatographic and electrophoretic techniques. Among these techniques, isoelectric focusing (hereinafter "IEF") has many attractive features.
The principle of IEF is based on the fact that certain biological materials (such as proteins, peptides, nucleic acids, and viruses) and even some living cells are amphoteric in nature--i.e., they are positively charged in an acidic media and negatively charged in a basic media. At a particular pH value, called the isoelectric point (hereinafter "pI"), these biomaterials will have a zero net charge.
Being charged in a pH gradient, the biomaterials migrate under the influence of an electric field until they reach the pH of their isoelectric point. At the isoelectric point, by virtue of their zero net charge, the biomaterials are not influenced by the electric field. Diffusion of "focused" biomaterials away from their pI will cause them to once again become charged, whereby they will electrophoretically migrate back to their pI. Thus, the biomaterials focus into narrow zones (defined by the pH of the medium and the electric field applied) from which the biomaterials can be selectively separated.
In one known method of isoelectric focusing, the pH gradient is established by the introduction of carrier ampholytes into the electric field. "Carrier ampholytes" are defined as ampholytes of relatively low molecular weight having conductance as well as buffer capacity, in the isoelectric state. Mixtures of synthetic polyaminopolycarboxylic acids have been used as carrier ampholytes.
In order to establish suitable pH gradients for IEF, it is necessary to have access to a great number of carrier ampholytes with isoelectric points well distributed along the pH scale. A commercial mixture of such amphoteric substances (called "Ampholine") is available from LKB Produkter AB, a Swedish Company. Ampholine is thought to be principally composed of polyaminopolycarboxylic acid molecules made by reacting polyamines with acrylic acid.
By manipulating the pH range of the carrier ampholytes, isoelectric focusing has the potential for high resolving power. However, the potential of isoelectric focusing as a means for separating amphoteric substances has not been realized because of the time necessary and the quality of separation of prior art processes.
Since acids are attracted to the anode of the electric field and bases to the cathode during electrolysis, an increasing pH gradient from the anode to the cathode will develop in a convection free electrolytic conductor. The success of isoelectric focusing depends on the satisfaction of three conditions: (1) that the pH gradient is stable in time; (2) that an electrolyte deficit does not develop within the field, thereby tending to quench the current and/or give rise to local overheating; and (3) that the pH gradient--d(pH)/dx--has a low value in the pH region of interest in the actual separation.
Isoelectric focusing is most often practiced in small-scale batch instruments where the fluid is stabilized by either gels or density gradients established by a nonmigrating solute such as sucrose. The capacity of such instruments for product separation is generally limited by the cross-sectional area of the apparatus. Because the apparatus cross-section is limited by the need to dissipate the heat generated by the electric field, larger scale preparative work has been proposed using continuous flow and recycling techniques.
One known technique which comes close to combining high resolution with large quantitative capacity is the recycling isoelectric focusing method disclosed in U.S. Pat. Nos. 4,204,929 and 4,362,612.
Currently known recycling isoelectric focusing (hereinafter "RIEF") techniques involve dividing a fluid containing carrier ampholytes into a plurality of reservoirs and passing the contents of the reservoirs through an isoelectric focusing cell. The isoelectric focusing cell separates the fluids from adjacent reservoirs with ion nonselective permeable membranes which allow interchange of fluid constituents from channel to channel, but which inhibit bulk fluid flow. Electrodes establish an electrical potential transverse to the fluid flow thereby creating a pH gradient between successive channels.
The fluid from each reservoir exiting the isoelectric focusing cell is pumped to the reservoir which feeds the isoelectric focusing cell. A heat exchanger cools the fluid within the reservoirs. As the fluid is pumped into the top of the reservoir, the fluid is directed from the bottom of the reservoir back into the isoelectric focusing cell.
This technique, however, has serious design flaws which preclude its routine use for both research and industrial scale application. The principal design flaw in this technique is that liquid containing material that is semipurified during each circular passage is immediately remixed with the crude starting material in the cooling reservoirs. Thus, the whole process constitutes a continual dilution process in which original crude mixtures in the reservoirs are continually diluted so that final purity is never truly achieved.
The constant remixing of crude and semipurified material in the prior art greatly increases separation time and compromises the resolution of the subcomponents and final purity of the isolated materials. In effect, an asymptotic dilution of contaminants occurs in each separation channel, and therefore a zero contamination level can never be achieved using currently known RIEF techniques.
This constant remixing of semipurified and crude material not only requires very long periods of time for attaining satisfactory degrees of separation, but also requires long periods of time to prefocus the carrier ampholytes into the initial pH gradient. Hence, the overall time required for prefocusing and actual separation is quite long.
Another serious drawback with the current RIEF techniques is the ability to dissipate the joule heat generated during the isoelectric focusing process. Current RIEF techniques cool the processed fluid within the reservoirs. Thus, extreme heating of the fluid sample may occur within the isoelectric focusing cell, irreparably damaging the desired biological sample, before the fluid can be cooled in the reservoirs.
In summary, the burgeoning genetic engineering market in the United States and other Western nations has created an acute need for high resolution protein purification techniques. The manipulation of human and animal genes for numerous hormones, enzymes, and other protein molecules into bacteria, yeast, or mammalian cell liner and the subsequent large-scale production of these proteins has created an acute need for rapid high resolution methods for purification of proteins and other similar biological substances. Although other techniques are frequently used as first or even second step techniques in the purification of these molecules, preparative isoelectric focusing in a narrow pH gradient is an ideal environment in which to do either initial or final stage protein purification.
From the foregoing, it will be appreciated that what is needed in the art are apparatus and methods for isoelectric focusing of amphoteric substances which combine high resolution separation with large quantitative sample capacity.
Additionally, it would be a significant advancement in the art to provide apparatus and methods for recycling isoelectric focusing of amphoteric substances which do not mix semipurified sample with the crude sample.
It would be another advancement in the art to provide apparatus and methods for isoelectric focusing of amphoteric substances which efficiently remove heat generated during the process, thereby permitting increased power input.
It would be a further advancement in the art to provide apparatus and methods for isoelectric focusing of amphoteric substances which rapidly separate the desired substance from accompanying impurities.
It would be yet another advancement in the art to provide apparatus and methods for isoelectric focusing of amphoteric substances which rapidly prefocus to establish a stable pH gradient.
Such methods and apparatus are disclosed and claimed herein.